Do I have to use coated tissue culture vessels for culturing PromoCell Normal Human Cells?

It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism, it is recommended to use the same coating material for a complete set of experiments. Cells that need to be grown on coated dishes: Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need FN- or VN-, or Collagen type I-coating (for details, please view the Product Manuals) when grown in PromoCell MSC Growth Medium XF (C-28019) and/or when being differentiated in our MSC Neurogenic (C-28015), Adipogenic (C-28016), or Osteogenic (C-28013) Differentiation Media. Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin- or Vitronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055, C-28056). For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020), the TC plates should be pre-coated with collagen type I.

Why are the cryopreserved macrophages abbreviated as hMDM-GMCSF(-) and hMDM-MCSF(-), respectively? What does the (-) stand for?

hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages . hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages . The macrophages can be seeded into all kinds of TC vessels. After plating, they can be maintained as biologically functional adherent cultures for several weeks.Optionally, user-customizable activation of the cells can be performed. For details, please see attached Application Note, page 2, Fig. 3 and page 5, Tab. 1).

What are the characteristic markers of HDLEC (C-12216, C-12217) and HDBEC (C-12211, C-12225)?

HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1). HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.

Technical library - RESOURCES

Q: Can you provide a list of cell lines that have been successfully used for tumorsphere formation with the PromoCell 3D Tumorsphere Medium XF?

The following cell lines have been tested at PromoCell to form tumorspheres in 3D Tumorsphere Medium XF (C-28070): U-87 MG MCF-7 MDA-MB-231 HT-29 HT1080 HepG2 A-549 Panc-1 LNCaP A-431 ⇒ For more details, please view the attached Application Note . In addition, we have received customer feedbacks for the following cell lines: HCT-116 (human colorectal carcinoma cell line) Capan-1 (human pancreatic adenocarcinoma cell line) PC3 (human prostate cancer cell line) C42B (osteotropic prostate cancer cell line) NCI-H23 (human lung epithelial adenocarcinoma cells) IMR-32 (human neuroblast cell line) A818-6 (human pancreatic ductal adenocarcinoma cell line) HEK293 (human embryonic kidney cells) Calu-1 (non-small-cell lung cancer cell line)

Usually 90-100% of the hMSC show a neuronal morphology after differentiation with our Mesenchymal Stem Cell Neurogenic Differentiation Medium (C-28015). 60-80% of them are positive for nissl bodies after a nissl stain.

Our hMSC-AT are characterized by their differentiation potential into chondrocytes, fat cells, and bone cells. In addition, we determine the presence of CD73, CD90 and CD105 expression as well as the absence of CD14, CD19, CD34, CD45 & HLA-DR expression by flow cytometry as proposed by the International Society for Cellular Therapy.

There are several factors that can influence successful transfections, e.g. viability and density of the cells, choice of the transfection reagent, quality and type of the transfected molecules (plasmids, siRNA, oligonucleotides), as well as the culture medium and supplements used.

When using PromoCell Endothelial Cell Growth Media for cell transfection, please follow the instructions below:

Heparin, which is included in our Endothelial Cell Growth Medium (C-22010/C-22110), Medium 2 (C-22011/C-22111) and Medium MV (C-22020/C-22120) may reduce the transfection efficiency. We therefore recommend to use our heparin-free Endothelial Cell Growth Medium MV2 (C-22022/C-22121). Alternatively, you may use Endothelial Cell Growth Medium Kit (C-22110), Medium 2 Kit (C-22111), or Medium MV Kit (C-22120) without adding the ECGS/heparin supplement to the Basal Medium.

Please note: Before and after transfection, the cells should be cultured in complete Growth Medium including heparin to ensure optimal growth.

It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism, it is recommended to use the same coating material for a complete set of experiments.

Cells that need to be grown on coated dishes:

Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need FN- or VN-, or Collagen type I-coating (for details, please view the Product Manuals) when grown in PromoCell MSC Growth Medium XF (C-28019) and/or when being differentiated in our MSC Neurogenic (C-28015), Adipogenic (C-28016), or Osteogenic (C-28013) Differentiation Media.
Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin- or Vitronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055, C-28056).
For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020), the TC plates should be pre-coated with collagen type I.

During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.

hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages.
hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages.

The macrophages can be seeded into all kinds of TC vessels. After plating, they can be maintained as biologically functional adherent cultures for several weeks.Optionally, user-customizable activation of the cells can be performed. For details, please see attached Application Note, page 2, Fig. 3 and page 5, Tab. 1).

The following cell lines have been

tested at PromoCell

to form tumorspheres in 3D Tumorsphere Medium XF (C-28070):

U-87 MG
MCF-7
MDA-MB-231
HT-29
HT1080
HepG2
A-549
Panc-1
LNCaP
A-431

⇒ For more details, please view the attached

Application Note

. In addition, we have received

customer feedbacks

for the following cell lines:

HCT-116 (human colorectal carcinoma cell line)
Capan-1 (human pancreatic adenocarcinoma cell line)
PC3 (human prostate cancer cell line)
C42B (osteotropic prostate cancer cell line)
NCI-H23 (human lung epithelial adenocarcinoma cells)
IMR-32 (human neuroblast cell line)
A818-6 (human pancreatic ductal adenocarcinoma cell line)
HEK293 (human embryonic kidney cells)
Calu-1 (non-small-cell lung cancer cell line)

Yes, we have received a customer feedback that our MSC Growth Medium 2 also works for rat MSCs. The rat cells grow nicely in this medium and have a good viability.

HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.

Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.

It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured, estrogen-responsive cells (Berthois et al., 1986). Furthermore, phenol red can also interfere with some analytical methods like photometric analyses.

Further Information

Berthois et al.; Proc Natl Acad Sci U S A. 1986 Apr;83(8):2496-500

The formulation of our basal media is proprietary information. If you need to know the concentration of a particular component for your experiments, please contact the PromoCell Technical Customer Service.